Method for real-time in-vivo synchronous monitoring of dual immunosuppression / activation and application

Abstract

The invention relates to the technical field of biological medicine detection, in particular to a method for real-time in-vivo synchronous monitoring of double immunosuppression / activation and application. The method provided by the invention is realized on the basis of the juvenile double-immune signal hybrid zebrafish at the age of 3-4 days, and can more accurately reflect the dynamic process and the mutual relation of the immune response of individuals and the effects of the dynamic process and the mutual relation in body health and disease occurrence and development. Furthermore, according to the method provided by the invention, commercial-grade strain hybridization is adopted, the animal model (less than or equal to 3 days) can be quickly constructed and obtained, and the experimental period is extremely short. According to the method provided by the invention, monitoring of synchronous and quantitative double immune cell inhibition / activation of the living body is realized; and the method is compatible with a standard fluorescence microscope (such as Tsai V16), and is suitable for popularization.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a method and application for real-time in vivo synchronous monitoring of dual immunosuppression / activation. Background Technology

[0002] In medical and biological research, the immune system is highly complex, encompassing multiple immune mechanisms and components. Real-time, in vivo, simultaneous monitoring of the inhibition / activation of different immune signals allows for a more accurate understanding of the dynamic processes, interrelationships, and roles of immune responses in health and disease development. For example, in tumor immunotherapy research, simultaneous in vivo monitoring of cellular and humoral immune responses to tumor antigens enables a more accurate assessment of treatment efficacy and changes in immune status. Currently, mammalian models cannot simultaneously monitor dual immune cell signals in vivo in real time; for instance, mouse models require sacrifice for data collection. Single-signal zebrafish models require multiple experiments (CN117129662A, detecting only T cells; CN117129661A, detecting only neutrophils); and the construction cycle for gene-edited dual-label zebrafish exceeds 6 months and requires advanced gene-editing technology, making it a high-barrier field.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method and application for real-time in vivo synchronous monitoring of dual immunosuppression / activation.

[0005] Specifically, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for real-time in vivo synchronous monitoring of dual immune suppression / activation, wherein the dual immunity is innate immunity and adaptive immunity; the method is implemented based on hybrid zebrafish juveniles with dual immune signals at 3-4 days of age.

[0007] Preferably, the innate immune signal indicators include at least one of neutrophil green fluorescence signal and macrophage green fluorescence signal.

[0008] Preferably, the signal indicators of adaptive immunity include T cell red fluorescence signal.

[0009] Preferably, the 3-4 day old dual-immune signal hybrid zebrafish juveniles are obtained by hybridizing a green fluorescent transgenic cell line and a red fluorescent T cell line.

[0010] Preferably, in the hybridization, the ratio of the male parent to the female parent is 1:1 to 2.

[0011] Preferably, juvenile fish 72–96 hours after fertilization are exposed to the test compound, and neutrophil and T cell signals are monitored simultaneously using a fluorescence microscope.

[0012] Preferably, the juvenile fish are treated with the solution of the test compound for 24-48 hours.

[0013] Secondly, the present invention provides the application of the method described in the first aspect above in drug screening and drug toxicity evaluation for non-disease diagnosis purposes.

[0014] Preferably, the drug is an anti-tumor drug, or a drug that alleviates the immunotoxicity of anti-tumor drugs.

[0015] Beneficial effects:

[0016] This invention provides a method and application for real-time in vivo synchronous monitoring of dual immune suppression / activation. This method is implemented using 3-4 day old hybridized zebrafish with dual immune signals, and can more accurately reflect the dynamic process, interrelationships, and roles of individual immune responses in health and disease development. Furthermore, the method uses commercial-grade strains for hybridization, enabling rapid construction of animal models (≤3 days) with a very short experimental cycle. The method provides in vivo synchronous quantitative monitoring of dual immune cell suppression / activation; and is compatible with standard fluorescence microscopes (such as the Zeiss V16), making it suitable for widespread application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0018] Figure 1 This is a schematic diagram of the hybridization screening of double-positive embryos according to the present invention.

[0019] Figure 2 This is a dual-signal suppression diagram of tartaric acid vinorelbine treatment according to the present invention. Detailed Implementation

[0020] This invention provides a method and its application for real-time in vivo simultaneous monitoring of dual immunosuppression / activation. This method is a simultaneous immunosuppression / activation evaluation method based on dual-fluorescence hybridized zebrafish juveniles. Specifically, it utilizes green fluorescent Tg (mpx:GFP) and red fluorescent TgBAC (cd247:mCherry) hybridized F1 generation zebrafish juveniles to simultaneously and quantitatively evaluate the suppression / activation of innate immunity (e.g., neutrophils, macrophages) and adaptive immunity (T cells). This method can collect both signals from the same fish, thus simultaneously and quantitatively evaluating the activation of both innate immunity (e.g., neutrophils, macrophages) and adaptive immunity (T cells), and simultaneously understanding the interaction and correlation of the two immune cells at the individual level. This invention also provides the application of the above method in drug screening and toxicity evaluation.

[0021] In a more specific embodiment, the technical solution provided by the present invention is as follows:

[0022] A method for simultaneously evaluating immunosuppression includes the following steps:

[0023] (1) Provide transgenic zebrafish fry, which are F1 generation obtained by crossing a green fluorescent transgenic cell line with a red fluorescent T cell line.

[0024] (2) Expose juvenile fish 72-96 hours after fertilization to the test compound.

[0025] (3) The number of neutrophils (GFP signal) and T cells (CY3 signal) were simultaneously detected by fluorescence microscopy.

[0026] (4) Calculate the Co-inhibition Index (CSI). When CSI ≤ 15%, it is determined to be a dual-pathway co-inhibition.

[0027] Preferably, in step (2), the juvenile fish are treated with an immunosuppressant solution for 24-48 hours.

[0028] Preferably, in step (3), the exposure time of the GFP channel of the fluorescence microscope is 1000-3000ms, and the exposure time of the CY3 channel is 700-3000ms.

[0029] Preferably, the detection sites are the posterior circulation (PCH) region and the thymus. Neutrophils are counted in the PCH region, and T cells are counted in the thymus region.

[0030] The method provided by this invention has significant advantages over traditional methods: (1) In terms of construction cycle, the animal model construction cycle of this invention is only 3 days, which is more than 98% less than the construction cycle of 3 to 6 months of traditional methods. (2) In terms of single detection cost, the single detection cost of this invention is <$10, which is 98% less than the single detection cost of >$500 of traditional (mouse) methods. (3) In terms of throughput, the detection throughput of this invention can reach 200 samples / day, which is 20 times higher than the throughput of 10 samples / day of traditional methods. (4) In terms of data dimension, this invention provides simultaneous monitoring of dual-pathway synergistic effects, which is a revolutionary improvement over the single immune index detection of traditional methods.

[0031] The aforementioned method for simultaneous evaluation of immunosuppression can be applied to screening immunomodulatory drugs, such as: high-throughput screening of the immunotoxicity of antitumor drugs; and evaluation of the immunosuppressive risk of natural products, health foods, and new food ingredients.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0036] In the following examples: Neutrophil inhibition rate of the data analysis model = [1 - (number of GFP+ cells in the experimental group / control group)] × 100%; T cell inhibition rate = [1 - (fluorescence intensity of T cells in the experimental group / control group)] × 100%.

[0037] In the following examples, the hybrid parents were all from commercially available sources, such as Cyclotech.

[0038] Example 1

[0039] This embodiment provides a method for preparing and verifying the F1 generation of hybrids.

[0040] (1) Origin of strain:

[0041] Commercially available green fluorescent transgenic cell lines and red fluorescent T cell lines.

[0042] (2) Parental ratio in hybridization:

[0043] Tg(mpx:GFP)♂×TgBAC(cd247:mCherry)♀=1:2.

[0044] (3) Embryo collection:

[0045] Incubate at 28.5℃ for 1 hour after fertilization.

[0046] (4) Selection and verification of juvenile fish:

[0047] GFP signal verification begins at 48 hpf, with samples showing signal expression and an intensity greater than 2500 ± 500 AU reserved.

[0048] CY3 signal was validated at 72 hpf, with the region located in the thymus and a signal intensity of 1850 ± 200 AU.

[0049] Example 2

[0050] In this embodiment, the F1 hybrid obtained in Example 1 was used to evaluate the immunotoxicity of the antitumor drug (vinorelbine tartrate).

[0051] (1) Experimental design: Drug: vinorelbine tartrate (0-100μM); Control group: physiological saline (n=30); Treatment time: 40 hours (72hpf→112hpf).

[0052] (2) Imaging parameters are shown in Table 1:

[0053] Table 1

[0054]

[0055]

[0056] (3) The results are shown in Table 2:

[0057] Table 2

[0058] Concentration (μM) T cell suppression rate (%) Neutrophil inhibition rate (%) CSI (%) in conclusion 18.53 21.9593 17.0247 4.9346 Synergistic inhibition 27.80 39.2262 29.2561 9.9700 Synergistic inhibition 37.06 43.0799 36.3884 6.6914 Synergistic inhibition 46.33 58.6342 37.6860 20.9483 Non-cooperative inhibition

[0059] The results in Table 2 demonstrate that vinorelbine tartrate induces synergistic immunosuppression at a certain concentration.

[0060] Example 3

[0061] This embodiment uses the hybrid F1 generation obtained in Example 1 to monitor the effect of broken-cell wall Ganoderma lucidum spore powder on alleviating the immunotoxicity of antitumor drugs.

[0062] (1) Model construction: Vinorelbine tartrate was injected into the circulatory system of F1 juvenile fish at 72 hpf. The control group was injected with physiological saline.

[0063] (2) Dynamic monitoring: Time points were selected at 24 and 48 hours. The detection indicators were: neutrophil count (GFP+ cells in PCH region) and T cell fluorescence intensity (thymocytes).

[0064] (3) Monitoring results are shown in Table 3 and Figure 2 :

[0065] Table 3

[0066] neutrophil count T cell fluorescence intensity (FU) Normal group 24 51.60±1.8330 228.82±4.6064 Model group 24 55.50±1.9791 194.12±4.5490## Sample group 24 51.30±1.2776 225.57±6.7817** Normal group 48 67.20±1.1624 365.50±15.1266 Model group 48 36.90±0.9597### 200.74±8.7505# Sample group 48 43.00±1.1643*** 233.07±6.9197*

[0067] Table 3 shows that the broken-cell wall Ganoderma lucidum spore powder can significantly increase T cell activity within 24 hours and simultaneously alleviate the immunotoxic effects of antitumor drugs within 48 hours (taking neutrophils and T cells as examples).

[0068] Example 4

[0069] This embodiment uses the hybrid F1 generation obtained in Example 1 to evaluate the effect of Cordyceps sinensis capsules in alleviating the immunotoxicity of antitumor drugs.

[0070] (1) Model construction: Vinorelbine tartrate was injected into the circulatory system of F1 juvenile fish at 72 hpf. The control group was injected with physiological saline.

[0071] (2) Detection after 48 hours. The detection indicators are: neutrophil count (GFP+ cells in PCH region) and T cell fluorescence intensity (thymocytes).

[0072] (3) The test results are shown in Table 4:

[0073] Table 4

[0074] Group T cell fluorescence intensity (FU) neutrophil count normal control group 414.68±12.8019 56.00±2.2998 Module group 200.29±9.8577 21.60±1.8086 500 μg / mL 337.57±18.3513 34.50±2.1512 250 μg / mL 256.12±20.9413 40.90±1.9802 125 μg / mL 284.86±18.2485 40.80±1.1813

[0075] Table 4 shows that Cordyceps sinensis capsules can significantly alleviate the immunotoxic effects of antitumor drugs.

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for real-time in vivo synchronous monitoring of dual immunosuppression / activation, characterized in that, The dual immunity refers to innate immunity and adaptive immunity; the method is implemented based on hybrid zebrafish juveniles with dual immune signals at 3-4 days of age.

2. The method according to claim 1, characterized in that, The innate immune signal indicators include at least one of neutrophil green fluorescence signal and macrophage green fluorescence signal.

3. The method according to claim 1, characterized in that, The signaling indicators of adaptive immunity include T cell red fluorescence signal.

4. The method according to any one of claims 1-3, characterized in that, The 3-4 day old dual-immune signal hybrid zebrafish juveniles were obtained by crossing a green fluorescent transgenic cell line and a red fluorescent T cell line.

5. The method according to claim 4, characterized in that, In the hybridization, the ratio of the male parent to the female parent is 1:1 to 2.

6. The method according to claim 5, characterized in that, Young fish were exposed to the test compound 72–96 hours after fertilization, and neutrophil and T cell signals were monitored simultaneously using a fluorescence microscope.

7. The method according to claim 6, characterized in that, The juvenile fish were treated with the solution of the test compound for 24-48 hours.

8. The application of the method according to any one of claims 1-7 in drug screening and drug toxicity evaluation for non-disease diagnosis purposes.

9. The application according to claim 8, characterized in that, The drug is an anti-tumor drug, or a drug that alleviates the immunotoxicity of anti-tumor drugs.

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